Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), one of the oldest known human maladies, is still is one of the major causes of mortality, as two million people die each year from this disease. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines, drugs and more specific and rapid diagnostics. The availability of the complete sequence of the Mtb genome and the use of new genetic and molecular methods has provided much new information concerning Mtb. The goal of the research described in this proposal, using this new information and methodology, is the identification of new targets in Mtb that will aid the development of these sorely needed anti-tubercular agents. The first period of research supported by grant AI-44856 was largely concerned with identifying Mtb genes and the proteins they encode that were potentially important in virulence. In the next grant period, we will investigate the roles in virulence played by genes that are induced in macrophages. We will also continue our studies on the sigma factors SigE and SigB and begin studies on SigL, concentrating on the genes that are transcribed by RNA polymerases containing these sigma factors, some of which, requiring sigma E, are necessary for Mtb virulence. The mechanism of regulation of sigma E and sigma L function by specific anti sigma factors will also be studied. We have shown that IdeR is the major regulator of Mtb iron flux. Since iron is essential for Mtb survival and virulence, we will identify and characterize iron/IdeR repressed genes and proteins they encode that form the iron acquisition machinery. IdeR is an indispensable protein, as its structural gene cannot ordinarily be inactivated, and we have shown that IdeR is also a positive regulator of iron storage genes. We plan to investigate the mechanism by which this protein activates gene expression, as this may help explain its essential nature.